The Extracellular Matrix: At the Center of it All

Stephanie L.K. Bowers, Indroneal Banerjee, and Troy A. BaudinoaTexas A&M Health Science Center College of Medicine, Division of Molecular Cardiology, Temple,TX 765

Keywordsextracellular matrix (ECM); cardiac function; cardiomyocytes; cardiac fibroblasts; endothelial cells;cardiac development; cell heterogeneity

IntroductionResponding to stimuli is a hallmark of the definition of life, and we are constantly adapting toour surrounding environments. Individual cells also read and respond to various signals thatsurround them. Communication between cells is essential, and is profoundly influenced by theextracellular matrix (ECM). In addition to providing structural support, the ECM contributesto individual cellular and collaborative organ-level functions. In cardiac physiology, the ECMfacilitates mechanical, electrical and chemical signals during homeostasis and thedevelopmental process, as well as in response to physiological stress or injury. These signalsmodulate cellular activities and interactions such as cell proliferation, migration, adhesion andchanges in gene expression during homeostasis and development. Furthermore, during post-myocardial infarction (MI) or hypertensive remodeling, components of the ECM can playagonistic and antagonistic roles that may contribute to progression towards a healthy recoveryor heart failure. This paradoxical relationship and the array of distinct cellular and acellularsignals that are involved in cell-ECM communication have made research in this area attractiveat both the molecular and cellular levels. This review discusses several developmentalprocesses that illustrate how cell-ECM communication is vital for proper cell-cellcommunication and cell behavior in the development of the heart, and how some of the sameprinciples apply to cardiac disease and pathological remodeling. Because of the large degreeof crosstalk between spatiotemporal and context-dependent signals of the ECM, it is imperativethat we approach research involving cell-ECM communication collaboratively, and interpretresults from as many angles as possible. We will discuss areas of focus that should helpelucidate the cellular mechanisms involved in cell-cell and cell-ECM interactions in the heart,and likely other organ systems as well.

Studies involving the complexity of the ECM in the heart are directly applicable to a host ofother physiological conditions. The cellular relationships and ECM factors discussed here areapplicable to a wide range of physiological conditions that must be considered when studyingany one system. Factors involved in the wound healing process, such as scar formation,

© 2009 Elsevier Ltd. All rights reserved.aTo whom correspondence should be addressed: Troy A. Baudino, Texas A&M Health Science Center College Of Medicine, Divisionof Molecular Cardiology, 1901 South 1st Street, Bldg. 205, Room 1R24, Temple, Texas 76504, USA., tbaudino@medicine.tamhsc.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptJ Mol Cell Cardiol. Author manuscript; available in PMC 2011 March 1.

Published in final edited form as:J Mol Cell Cardiol. 2010 March ; 48(3): 474–482. doi:10.1016/j.yjmcc.2009.08.024.

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collagen turnover, matrikines and cellular migration, are also found in cardiac developmentand response to injury [1–3]. In cancer progression and metastasis, epithelial-mesenchymaltransition (EMT)-like transformations and interactions among tumor and stromal cells aresimilar to the cellular and acellular milieu seen in the heart [2,4]. Likewise, the basis of anappropriate immune response involves a conglomerate of autocrine, paracrine and hormonalsignals, which are differentially released by particular cells, and include spatiotemporalreceptor-ligand interactions and expression, just as in cardiac development and adult function.Furthermore, research pertaining to obesity and diabetes, vascular biology and the nervoussystem offer other areas of crossover, as patients with disorders in these areas present withconditions sufficient to alter homeostatic and pathologic cardiac function.

The Malleable ECMIt is important to regard the truly dynamic nature of the ECM and the variety of functions it isrequired to serve. Numerous studies reveal differences in the presence of specific ECMcomponents during distinct physiological states (Figure 1). The diversity of the ECMcomposition is illustrated by the unique environments that are required for cell survival andproper function in the developing heart, a normal adult heart and a heart under pathologicalstimulation. Each cellular environment is subject to a host of autocrine and paracrine signalsthrough cytokines, growth factors and hormones. Moreover, the different microenvironmentswithin the developing or adult heart present with unique biochemical niches, largely driven bycell-cell and cell-ECM interactions.

Early cardiac development is largely dependent upon genetic integrity (i.e. mutations and DNAdamage) and more immediate environmental signals (i.e. maternal hormones, mechanical andchemical signaling). Conversely, the adult heart is subject to a multitude of circulating factorsand reciprocating signals from other organ systems within the body, especially in response topathological conditions (i.e. from the kidneys, the central nervous system and the immunecompartment). The presence of these “external” signals could alter the sensitivity of themyocardium and cardiac vasculature to more proximal signals from the surrounding cellularenvironment, during both homeostatic and pathological conditions. Although discussion ofcirculating factors and other organ systems are not within the scope of this review, they are animportant element of the cellular microenvironment of the heart.

The ECM in the Developing and Adult HeartDuring cardiac development, there is an array of environmental signals that ensures that cellmigration and transformation occur in a specific spatiotemporal order. These signals includechemical and mechanical signals originating from both the cells and the ECM. During neonataldevelopment, the ECM constitutes a thin layer within the epicardium, where epicardialmesenchymal cells and ECM thickening (collagen deposition) arise concomitantly [5]. Theseobservations coincide with the differential functions of the fetal and postnatal heart, whereventricular wall thickness and tensile strength (stiffness) increase to accommodate changes infunctional requirements following birth and the opening of the ductus arteriosis [6]. In addition,the developing heart tube is arranged as an endothelial cell (EC) layer surrounded bymyocardial cells, with an acellular matrix composed of proteoglycans, collagens andglycoproteins found in between the inner EC layers [7]. This matrix serves to maintain tubestructure and proper regulation of flow during embryologic heart development.

The composition of the ECM is a major factor in the process of EMT, which occurs duringnumerous developmental stages, as well as in response to pathology [4,8]. EMT is critical fororganogenesis, specifically during cardiac cushion morphogenesis and valve formation.Secretions from myocytes and the surrounding ECM stimulate ECs to transform and invadethe surrounding mesenchyme and ECM [7,8]. One component of myocyte (and EC) secretions

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that promotes EC migration and EMT are members of the transforming growth factor-β (TGF-β) family of cytokines and their receptors [7]. Signaling via the TGF-β type 1 receptor, Alk5,seems to be essential for normal EMT and function of the epicardium during cardiacdevelopment [9]. Interestingly, Alk5 signaling is not essential for proper development of themyocardium, suggesting signal compartmentalization.

Early in cardiogenesis, the mouse heart tube is formed from endocardial and myocardial celllayers that surround the ECM. The heart tube subsequently loops around, and specific heartregions begin to develop into chambers [10]. Embryonic cushions, which proliferate anddevelop into the primordial heart valves following heart looping, differentiate from ECs inresponse to an increase in targeted, localized ECM synthesis by AV canal myocytes, which isthen followed by an increase in ECM degradation as the cells undergo EMT and migratethrough the mesenchyme to develop into cushions [11]. Because of their fibroblastic nature,these cushions are effectively formed from a localized expansion of the ECM. Only certaingroups of cells within the heart tube respond to the paracrine and autocrine signals necessaryfor the initiation of cushion formation, and subsequent differentiation into a fibroblasticphenotype for valvulogenesis [12]. As mentioned, TGF-β is critical for proper EMT during allstages of development, including cushion formation and differentiation.

Another group within the TGF-β family of proteins that are critical for cushion differentiation,as well as subsequent valve formation, are the bone morphogenic proteins (BMPs). DifferentBMP proteins are expressed in specific regions of the developing heart at specific times [10].Interestingly, BMPs can be retained within the ECM until subsequent activation, which makesit difficult to determine the origin of such signals. Because of their spatiotemporal expression,TGF-β and BMPs are considered possible inducers of cushion formation and differentiation[10]. Periostin, which will be discussed later in this review, is another ECM protein that playsa key role in EMT and cushion formation, as well as differentiation into the fibroblasticphenotype [12,13].

The organization and composition of the ECM is also critical to cardiac valve developmentand function. Following heart looping and the initiation of unilateral blood flow, endocardialcushions are formed from a localized expansion of ECM [14]. The proper transformation of aspecific fraction of ECs, and their reception of signals from the surrounding mesenchyme andECM, is critical for appropriate valve leaflet formation and subsequent valvulogenesis [14].The composition and integrity of the ECM mediates critical cellular migration andcompartmentalization that affects cardiac structure formation and heart function. The ECM isalso integral to the spatiotemporal process of condensation and increased density ofmesenchymal cells within valve leaflets [15]. Areas of condensation within the developingheart leaflet vary with embryonic and fetal stage, where the ECM factors involved (i.e.fibronectin and N-cadherin) characterize areas involved in condensation. The spatiotemporalexpression (or ECM release) of BMPs also plays a role in valvulogenesis. Although traditionalknockouts of single BMP proteins reveal no major cardiac defect, double-mutants result insevere cardiac deficiencies, and have enabled the investigation of these proteins in varioustissues throughout development. A deficiency in BMP receptor type II (BMPRII), eitherthroughout the embryo or localized to the endocardium, results in severe valve cushion andseptal defects, as well as abnormal remodeling and positioning of the aorta [16].

The adult mammalian heart consists of a network of different cell populations that cooperateto maintain heart function under normal and pathological conditions. Structurally, theventricular myocardium is organized into laminae of myocytes approximately 2–5 cells thick.These layers of myocytes are surrounded by an endomyosial collagen network, where cardiacfibroblasts interconnect with myocytes, and endothelial cells and vascular smooth muscle cellsare largely confined to the coronary vasculature [17]. Resident stem cells and ‘transient’

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circulating cells, such as platelets, monocytes and mast cells are found throughout themyocardium and interstitial space. In addition, the presence of a supportive fibrillar, connectivenetwork involved in cardiac cell physiology has been observed since the late 1970’s [18]. Thisnetwork is a critical component of the development and maintenance of cellular organization.The interconnections among cells and between cells and the ECM play critical roles in thephysiology of the heart, as communication between different cardiac cell populations,specifically myocytes, fibroblasts and ECs, is important for proper heart function. Differencesin the composition of the ECM, such as collagen content or enzyme expression, are directlyinvolved in cell adhesion, migration and overall cellular communication.

The ECM is a dynamic collection of macromolecular proteoglycans, glycoproteins, proteases,collagens, growth factors and cytokines; modulation of these various components facilitatesproper mechanical, chemical and electrical signaling between cells [19]. The signals providedby the ECM are either active or latent, and are critical in homeostasis and pathologicalremodeling. The different cell populations of the heart vary in their ability to make and secretedifferent ECM components (Figure 2). Generally, fibroblasts and vascular smooth muscle cells(VSMCs) are involved in fibronectin and collagen type I and III production, and VSMCs,myocytes and ECs produce type IV collagen and laminins [20]. Cardiac myocytes also producecollagen VI and proteoglycans [21]. Additionally, cardiac fibroblasts are the primary producersof matrix metalloproteases (MMPs) [19,22] and periostin [23], and also secrete variousparacrine and autocrine factors, such as interleukin-6 (IL-6), TGF-β, endothelin-1 and tumornecrosis factor-α (TNF-α) [19]. Cardiac fibroblasts are sensitive to mechanical stimulation,depending on the environment provided by myocytes and the surrounding matrix [24]. TheECM composition and organization, along with the cellular components, affects forcegeneration, receptor expression and chemical signaling which are important for proper cardiacfunction. Interestingly, many genes that are no longer expressed once development is completeare often re-expressed during remodeling, such as after MI [2]. Normally, most ECM proteinsare primarily produced by the cardiac fibroblast; however, in pathological states such as MI,myofibroblasts, neutrophils, mast cells and macrophages also produce ECM proteins.

ECM in Cardiac Pathology and RemodelingIn response to MI, the process of cardiac remodeling includes angiogenesis, myocytehypertrophy and fibroblast proliferation, which results in increased collagen deposition andalterations in ECM proteins and intracellular hormones, cytokines, matrikines and growthfactors. These factors include IL-6, TGF-β, endothelian-1, TNF-α, fibroblast growth factor(FGF) and angiotensin II (Ang II) [19]. These chemical signals play roles in apoptosis,angiogenesis and cardioprotective responses following pathological insult [19]. Inflammationand scarring occurs at the site of injury, with a concomitant increase in laminins and β1-integrinat the myocardial interface of scar tissue [25]. Activated fibroblasts accumulate at the site ofinjury where they remain indefinitely, leading to an increase in local collagen deposition andfibrosis [3]. An imbalance in the synthesis and degradation of collagen and other ECM factorscontributes to myocardial pressure increases and cardiac dysfunction. Ang II and TGF-β aresecreted by fibroblasts, which alters cellular processes near and remote to the infarct, especiallycollagen turnover [3]. The secretion of TGF-β can trigger myocyte hypertrophy and reducecollagenase production, while leading to increases in tissue inhibitor of metalloprotease(TIMP) synthesis, which effectively decreases ECM degradation and leads to ECMaccumulation. Stiffening of the myocardium via fibrosis and increased deposition of collagensand other ECM proteins can alter the mechanical and electrical dynamics of heart function,leading to cardiac dysfunction.

Following cardiac injury, surviving cardiomyocytes alter their cell-cell and cell-ECMinteractions in response to surrounding cell death, which leads to ventricular remodeling [25].

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These alterations occur both adjacent and distally to the site of the infarct [26,27]. The ECMcan be temporarily remodeled, reversibly remodeled or fully adapt to the changes inbiomechanical load; however, too large of a load increase over a long period of time results indetrimental collagen deposition, left ventricular dilatation and heart failure [28]. Thecomposition and arrangement of collagen fibers within the heart determines the stressdistribution across myocytes and movement of fluid within the extracellular compartments.Myocytes are highly sensitive to mechanical force that is transduced into chemical signalsrequired for appropriate myofibrillar assembly and coordinated muscle contraction [29]. Dueto hypertrophic conditions, transmission of mechanical load throughout the myocardiumtriggers a cascade of cellular events. Depending upon the nature of mechanical stimulation,ventricular wall thickening and increased contractility can be either beneficial, such as duringexercise or development, or deleterious, as in response to pathophysiological signals [30]. Inthe latter condition, the heart undergoes maladaptive remodeling, such as fibrosis, possiblyresulting in cardiac failure.

Variability of the ECM Depending on Species and GenderOn the cellular side, the ratios of different cardiac cell populations, namely myocytes,fibroblasts, ECs and VSMCs, change throughout development and into adulthood [31]. Theseobservations are in accord with temporal relationships in alterations of ECMmicroenvironments of the heart. From heart tube formation to closure of the ductus arteriosis,as well as during cardiac development and pathological remodeling, changes in cellularphenotype confer reciprocating changes in ECM composition. However, although coordinatedwithin a system, the functions of the ECM and effector molecules involved are not alwaysconsistent between different species, and gender-specific differences in ECM physiology areapparent.

Changes in the ratios of different cardiac cell populations, namely cardiac myocytes andfibroblasts, occur during development [31] and adult remodeling [28]. However, the balanceof the different cell populations in the heart is species-specific in some instances, as studiesshow far more fibroblasts and fewer myocytes in the adult rat heart compared to the mouseheart, which is mostly composed of myocytes [31]. These differences can largely be explainedby changes in biomechanical load requirements through increased body mass and circulatorydemands. The law of Laplace states that the larger a vessel’s radius, the larger the wall tensionrequired to withstand a given internal pressure. Alteration in the radius of the heart in a trans-ventricular tension model designed to reduce the radius of curvature of the left ventricle hasbeen shown to alter hemodynamic function and wall thickness [32]. As the radius of curvatureof the left ventricle of a rat is larger than that in the mouse, this suggests that the wall tensionon the heart would be increased in the rat when compared to the mouse. This suggests that therat would need an increased amount of connective tissue; therefore, this would require anincrease in the number of fibroblasts based on the radius of the heart given equal pressures, asit has been demonstrated that the mean arterial pressure in the mouse is almost identical to thatobserved in the rat. Thus, an increase in tension would require an overall increase in connectivetissue and interstitial cells. In accord with this hypothesis, previous studies demonstrate thatthere are increased numbers of cardiac fibroblasts in the rat compared with the mouse [31].Therefore, the conclusion is that the amount of ECM in the heart would be greater in the ratthan in the mouse, indicating an increase in tension on the heart. This has been shown in studiesassaying for hydroxyproline content in the heart [33]. In addition, previous studies from ourlaboratory have demonstrated that the adult rat heart contains more hydroxyproline comparedwith the mouse, 9.36 versus 5.95 µg collagen/mg dry heart wt, respectively [31]. Takentogether, these previous studies suggest that larger mammals, which all display similar meanarterial pressures, would have greater wall tensions given their increased radius of curvature.Indeed, previous studies have demonstrated an increase in hydroxyproline content as seen in

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a comparison between rats and dogs [34]. Thus, given that cardiac fibroblasts are the mainproducers of collagen in the heart, the rat heart may require more fibroblasts to provide themeans to compensate for the increase in wall tension.

Species differences in ECM factors are seen in other aspects of heart development andremodeling as well. For example, in mouse and canine models of myocardial infarct, similarpatterns of ECM deposition are seen following reperfusion; however, the time course ofremodeling and scar formation is significantly shortened in the mouse, likely reflectingdifferential gene and/or protein expression patterns [35]. These differences illustrate theimportance of dynamic cell-cell and cell-ECM interactions in responding to environmentalstimuli to ensure proper heart function. They also lend to differences in responses to injury, asdifferent signals would likely be required for healing processes in dissimilar cellularenvironments. The critical process of EMT and consequent cushion and valve formationrequires the spatiotemporal expression of different TGF-β isoforms, as well as differential EMTprogression, in avians when compared to mice [10]. Rather than ignoring these differences,we should further examine them in order better define the roles of a particular protein orpathway. Understanding the composition and signals of the ECM during remodeling could bebeneficial towards regeneration of tissue at sites of defects or wounds.

Also relevant to the current discussion is the gender specificity of cardiac function and responseto injury. Recent studies examining aortic valve stenosis demonstrated gender differences incytokine and ECM expression during ventricular remodeling [36]. Collagen I was upregulatedmore than any other ECM component in women, and different branches of the TGF-β signalingpathway were activated in women versus men. Aside from gaining insight into gender-specifictherapies, these studies ascertain that, in knockouts and other models of cardiac developmentand dysfunction, care should be exercised when combining data from male and female animalsubjects. Future studies should be focused on dissecting the functional roles of these pathwaysin the downstream responses to hormonal and mechanical factors.

Angiogenesis and the ECMFormation of blood vessels depends upon environmental cues that regulate EC function. TheECM acts as a physical scaffold for cells and a storage area for cytokines and growth factors.In addition, the mechanical and chemical properties of the ECM transmit information to cellsthat influences their behavior through interactions with cell surface receptors termed integrins,which are discussed in further detail below. During angiogenesis, the perivascular ECM playsa critical role in determining the proliferative, invasive and survival responses of ECs toangiogenic factors. Dynamic changes in the ECM, as well as in the vascular cells (ECs andVSMCs) act together to regulate new vessel formation. Digestion of ECM components byMMPs is essential for invasion by ECs, but also creates ECM fragments, which can antagonizethe function of integrins (Figure 3). Integrin interactions with ECM proteins potentiate thesignaling events that are critical to angiogenesis. Indeed, the signaling activity of many receptortyrosine kinases is dependent upon interactions between integrins and the ECM.

Besides interacting with the ECM, ECs interact with other cell types, including other ECs andpericytes. Additionally, we have observed interactions between cardiac fibroblasts and ECsboth in vitro and in vivo, suggesting that fibroblasts are also important for blood vesselformation during development and possibly during disease. As mentioned above, fibroblastsplay a role in the development, growth, and remodeling of tissues. They do so through synthesisand deposition of ECM, as well as through secretion of cytokines and growth factors, whichallow them to modulate their environment through autocrine and paracrine signaling. Inaddition, fibroblasts can also secrete vascular endothelial growth factor (VEGF). VEGF actson endothelial cells and is important for stimulating angiogenesis and coronary collateral

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formation for restoring the blood supply to injured myocardium. To date, few studies havedirectly investigated how fibroblasts contribute to blood vessel formation and this should bean intense area of investigation for years to come.

Other factors that have been shown to play a role in angiogenesis are also produced byfibroblasts, such as MMPs and TIMPs. MMPs can regulate EC proliferation, adhesion andsurvival leading to activation or inhibition of angiogenesis. MMPs act by degrading the ECM,which can promote EC migration and sprouting, or MMPs can cleave and release anti-angiogenic factors that inhibit these events. TIMPs have been shown to inhibit angiogenesis,as well as promote vessel formation. Thus, like MMPs, it appears that the function of TIMPsis context dependent. Recent studies from Lilly and colleagues demonstrated that TIMP-1 issecreted by fibroblasts and increases vessel formation [37]. In these studies, they observed thatactivities of TIMP-1 were MMP-dependent. In addition, they observed that direct interactionbetween fibroblasts and endothelial cells is necessary for optimal vessel formation [37]. Thesestudies underline the significance of fibroblast-derived factors and cell-cell interactions inregulating angiogenesis. Future studies need to be directed at defining the cell surfacemolecules that play a role in these cell-cell interactions, as well as the importance of the ECMin regulating this process, especially during remodeling following cardiac insult.

The ECM PlayersIntegrins

Integrins are essential for ECM interactions with cardiac myocytes and fibroblasts, and overallstructure and cell communication in the heart. They mediate cell-ECM and cell-cellcommunication in a bidirectional manner – that is, extracellular events affect nuclear activity,while signals that originate in the nucleus affect cell surface protein expression and function.Integrins are large transmembrane ECM receptors that consist of alpha and beta subunits andhave a large extracellular domain and small cytoplasmic signaling domain; splice variants andheterodimerizations of the subunits confer a wide range of ligands while also maintaining ahigh level of specificity [27]. They are critical for cell behavior, specifically cell migration andsurvival, during virtually all developmental stages, as well as pathological remodeling. Bothexternal and internal signaling can affect integrin expression and clustering within the cellmembrane, as well as integrin conformation and ligand affinity. The list of integrin ligands isexpansive [38]; most commonly, integrins are receptors for collagen, laminin, fibronectin andthrombospondin, as well as tenascin-c, osteopontin and periostin.

The temporal and spatial localization of integrins is altered during developmental progressionand normal heart function, and is highly coordinated with ECM composition. Their properexpression is necessary for the process of developmental and pathological hypertrophy in theheart. A large number of knockout studies have found both overlap in integrin function andspecific roles for particular integrin subunits and heterodimers, and are elegantly reviewed byRoss and Borg [27]. Knockouts of specific isoforms of β1-integrin result in poor blastocystdevelopment and implantation, embryonic death and altered perinatal heart function [27].Recently, β1-integrin was implicated in differential neonatal and adult cardiac fibroblastfunction [39]. In these studies, it was demonstrated that embryonic cardiac fibroblasts promotedmyocyte proliferation through the secretion of fibronectin, collagen and heparin-bindingepidermal growth factor, all of which are mediated by the β1-integrin receptor [39].Interestingly, these studies also showed that adult cardiac fibroblasts promoted myocytehypertrophy through β1-integrin receptors, indicating a functional switch in fibroblastphenotype and/or integrin activation. These studies illustrate the critical elasticity of cell-ECMinteractions, and how these interactions change in response to environmental demands. Incardiac remodeling following MI, an isotype switch from β1D to β1A integrin in myocytes hasbeen shown to be regulated by TNF-α during certain stages of the healing process [40],

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exemplifying the role of cytokines in the spatiotemporal regulation of integrins. In fact, theexpression of several cytokines are associated with integrin regulation, including TGF-β andangiotensin II [40]. As such, integrins may provide an important link between circulatingcytokines and/or growth factors and the response of cardiac cells to signals originating outsideof the heart. This is especially relevant to preventative therapy or in diseases where there is asubstantial risk for secondary heart disease, such as with diabetes.

A current molecule of interest that utilizes several integrin signaling pathways is integrin-linkedkinase (ILK). This molecule localizes at focal adhesions and areas of mitotic spindle formation,and can function as both a kinase and adaptor protein that regulates a number of cellularprocesses such as EMT, angiogenesis, cytoskeletal arrangement and mitosis [rev in 41]. Itlargely acts through the PI3K, β-catenin and E-cadherin signaling pathways, andoverexpression of ILK has been linked to cancer formation and progression. In fact, clinicaltrials are currently underway to examine how using ILK inhibitors as chemotherapeutic agentsmay attenuate cancer cell mitosis and cancer progression [41]. Further investigation of ILKand other adaptor and effector molecules will not only elucidate the specific pathways involvedin integrin signaling and expression, but also precise methods of crosstalk between integrinsand other signaling and cell survival pathways.

Fibronectins and LamininsSecreted by cardiac fibroblasts, fibronectin is an adhesive glycoprotein that associates withmultiple ECM factors, and whose substrate specificity is regulated by different splice variants[20,42]. Fibronectin is an essential component of vascular morphogenesis, the importance ofwhich is demonstrated by the embryonic lethality of fibronectin knockout mice [42]. Bothmechanical and chemical factors can induce fibronectin expression in development andcardiopathology, which is often preceded by an increase in collagen and TGF-β expression[20]. A glycoprotein found in the basement membrane of cardiac myocytes, laminin containsdifferential binding domains for several ECM components, including collagens and cellmembrane receptors [21]. Laminin is largely produced by cardiac myocytes, VSMCs and ECs[20,21]. Knocking out various subunits of laminin and laminin receptor isoforms results inaltered cardiac development and/or adult cardiac function [21]. As mentioned above, the ECMplays a critical role in angiogenesis and laminin may be a critical player in this process.

CollagensCollagens are an indispensible component of ECM. Different types of collagen exhibit uniqueproperties, such as tensile strength and fibril formation [28,43]; therefore, its synthesis anddeposition is highly coordinated. Collagen deposition can be regulated by autocrine andparacrine hormones, such as Ang II, TGF-β, aldosterone and angiotensin-converting enzyme(ACE) [3]. In response to particular biomechanical and biochemical signals, cardiac fibroblastsincrease collagen synthesis and deposition. Fibroblast secretion of Ang II stimulates theexpression of TGF-β and collagen I synthesis [44]. Moreover, Ang II can increase cytokineproduction in myocytes and enhance fibroblast sensitivity to cytokines [44]. ACE inhibitorsdecrease TGF-β, fibrosis and collagen deposition, and improve outcome hypertrophy andfibrosis [21]. One mechanism of action seems to be through the inhibition of hydrolysis of N-acetyl-seryl-aspartyl-lysl-proline (Ac-SDKP) by ACE, which decreases fibroblastproliferation and collagen synthesis [45]. Ac-SDKP, in turn, has been shown to reduceGalectin-3 expression, which is an endogenous lectin involved in inflammatory cellrecruitment and increased cytokine secretion, fibroblast proliferation and ventriculardysfunction. The inhibition of Galectin-3 results in reduced fibroblast proliferation, collagendeposition and inhibited macrophage activation and migration, leading to a decrease in cardiacdysfunction following Galectin-3 treatment [45]. In addition, chronic activation of the renin-angiotensin system is associated with the presence of inflammatory cells and fibroblasts that

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precede changes in the vasculature and affect cell recruitment and collagen deposition, leadingto fibrosis [3].

Some collagens are only expressed during specific stages of development or remodeling,depending on the required function of collagen expression, and genetic defects in differentcollagens generally results in pathologic heart valve structure and dysfunction [43]. Varioustypes of collagen are found in different regions of the heart depending on the stiffness andcontractility required for tissue function [43]. As different collagens are used for specificfunctions, the ratio of collagen types deposited in certain areas of the heart contributes to tissueflexibility and function; a balance of collagen degradation and synthesis is necessary, but canbecome maladaptive following injury if stiffness is exacerbated [28]. It is important to notethat an increase in collagen deposition, per se, is not the only cause for collagen-induceddecreased contractility in response to cardiac injury or hypertrophy, as maladaptive changesin the ratios and type of collagen expressed will have the same pathologic affect. In additionto these alterations in collagen deposition following hypertrophy, changes in the degree ofcollagen crosslinking have been associated with altered cardiac function [rev in 28]. Thearrangement of the collagen network can affect the conductance of electrical signals andbridging of myocytes by cardiac fibroblasts [17]. During normal electrical conduction, cardiacfibroblasts and myocytes interact with each other and themselves via homotypic andheterotypic cell-cell signaling through connexins (Cx 40, 43 and 45) [17,46]. Connexins arecritical components of intercellular communication and cellular adhesion that mediateelectrical conductance, among other cell-cell interactions. As fibroblasts themselves are notelectrically excitable but are primary producers of ECM [19], they contribute to cardiacmyocyte function through avenues such as connexin receptor expression (signal diffusion) andcollagen deposition (signal inhibition) [46]. Overall, myocyte-fibroblast coupling andincreased collagen deposition have been shown to decrease electrical conduction velocity[47]. The ability to control collagen deposition by cardiac fibroblasts in a spatiotemporalmanner, which would include ECM contributions, would target myocardial development,wound healing, cardiac fibrosis and a host of consequent disorders. Along the same lines,approaches for intervening in collagen metabolism need further study for potential preventativeand therapeutic intervention strategies.

PeriostinAnother secreted ECM protein, periostin (or osteoblast-specific factor 2), is a 90kDa proteinthat belongs to the fasciclin family of genes (with fasciclin-1, bIG-H3, stabling-1 and -2), andis able to bind to several integrin heterodimers [11]. Periostin is most often secreted byfibroblasts or cells that acquire a fibroblast-like phenotype during development and followinginjury [23]. Periostin can directly interact with other ECM proteins, including collagen I,fibronectin and tenascin-c, as well as several integrins. Periostin-deficient mice display grossabnormalities in cell differentiation and valve formation, possibly through a TGF-β-dependentpathway [6], which illustrates a regulatory role of periostin in cardiac morphogenesis [18]. Inthe developing chicken and mouse, periostin is localized to the endothelium of the ventriculartrabeculae and in the endothelium and mesenchyme of the outflow tract and AV cushions[48]. Later in cardiac development, periostin is expressed in the fibrous region of the heart,suggesting that shear stress and altered hemodynamic load affect its expression [48,49]. Overthe course of embryonic development, periostin expression is observed at E10.5 and steadilyincreases until E14.0–17.0, an expression pattern that mimics that of the endocardial cushionto valve formation [50]. Indeed, recent studies have demonstrated that loss of periostin in miceresults in valve defects and a marked reduction in postnatal survival [49]. In the developingheart, periostin is absent from cardiomyocytes, but is expressed in non-cardiomyocytes,specifically fibroblasts and VSMCs [51]. Following vascular injury, periostin expression isobserved from VSMCs and has been shown to be associated with VSMC differentiation in

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vitro and promote cell migration [52]. Interestingly, periostin expression is not regulated byFGF in VSMCs, which may useful in delineating smooth muscle and fibroblast lineages.

In addition to its role in cardiac morphogenesis and development, periostin’s involvement incardiac remodeling is under intense investigation. In adult mice, overexpression of periostinvia adenoviral administration results in ventricular dilation, a decrease in myocytes, increasedcollagen deposition and decreased myocyte spreading and fibroblast adhesion [23]. Manyreports have demonstrated a critical role of periostin in developmental and adult ECMremodeling, collagen fibrillogenesis, and overall cardiac hypertrophy and remodeling [18]. Werecently reported that IL-6 knockout mice have an increased fibroblast to myocyte ratio,collagen deposition and cardiac dilatation that was accompanied by a marked increase inperiostin expression [53]. Although a direct causal effect of IL-6 on periostin cannot beconcluded from these studies, it illustrates direct and/or indirect links between cell populations,ECM composition and different signaling factors.

Animals that undergo pressure overload hypertrophy show a significant increase in periostinexpression. Upregulation of periostin via the PI3K pathway has been demonstrated, and leadsto the induction of TGF-β, FGF and Ang II [13]. Other studies have confirmed the involvementof PI3K pathways during cardiac remodeling, even in the absence of periostin signaling, whichhas been demonstrated in both embryonic development and the adult heart [6,54]. Furthermore,loss of anti-hypertrophic factors such as atrial natriuretic peptide (ANP) results in an increasein periostin expression of 20–40-fold when compared to control mice. However, this effectwas only seen in ANP knockout mice that had pressure overload hypertrophy, not in mice thatwere ANP-deficient without hypertrophy [55]. Taken together, these studies suggest thatincreased hemodynamic stress plays a role in the expression of periostin, and that periostinplays a critical role in the pathology of the heart.

While periostin clearly plays an important role in cardiac development and response tohypertrophy, the specific mechanisms by which it contributes to these processes are currentlyunder debate. A recent study implicated the expression and secretion of periostin withactivation of DNA synthesis and cellular proliferation in rat cardiac myocytes [54]. However,in another study, Molkentin and colleagues did not observe an increase in cardiomyocyteproliferation in response to periostin treatment [56]. While results from these studies arecontradictory, they underscore the importance in understanding the role of periostin in theheart, given that induction of cardiomyocyte proliferation has serious implications in thetreatment of patients following myocardial injury. Periostin has many clinical implications andfurther investigation into the ability of periostin to initiate EMT could be useful inbioengineering/in vitro systems. The ability of periostin to reduce cardiac scar formationfollowing ischemia or hypertrophic insult could also be further examined.

MMPs/TIMPsWhile MMPs and TIMPs are expressed by virtually all cell types in the heart [57], fibroblastsare the main contributors. Various interactions among MMPs and TIMPs affect theirubiquitination and degradation, induce signaling cascades through dimerization and alter geneexpression and cellular localization, effectively altering the extracellular environment topromote or hinder cell signaling, migration and survival. MMPs and TIMPs have beenimplicated in an array of physiological responses following cardiac hypertrophy, and specificchanges in left ventricular function have been correlated with MMP expression [58]. FollowingMI in rats, different MMP and TIMP isoforms are activated in a coordinated fashion duringspecific stages of remodeling [59], contributing to the spatiotemporal regulation of ECMsignaling.

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In the heart, MMP-7 is expressed by macrophages and myocytes [60], and appears to influencecleavage of Cx43, a critical factor in gap junction-mediated cell communication and electricalconduction involved in cardiac remodeling. Altered Cx43 expression, cardiac remodeling andsurvival have been observed in MMP-7 knockout mice following myocardial infarction [60].These studies leave open the possibilities of MMP inhibitors or even focusing on thedownstream substrates of MMPs as a therapeutic option following cardiac injury.

MatrikinesThe term matrikine has been proposed to define those circulating and tissue-specific peptidesthat are produced by the partial proteolysis of ECM macromolecules, and are able to affectcellular function. Once cleaved, matrikines are free to interact with cell surface receptors,thereby affecting a wide array of biological processes including matrix degradation, celladhesion, migration, proliferation, apoptosis and protein synthesis [61]. Examples ofmatrikines are those peptides derived from elastins, connective tissue glycoproteins, lamininsand collagens. Additionally, matricryptic sites are thought to exist on insoluble ECM moleculesin tissues, matricellular ECM proteins and plasma-derived ECM molecules [62]. These sitesonly refer to those not normally exposed in the intact, activated peptide. Current researchinvolving matrikines is largely targeted toward their protective actions in tumor-stromalinteractions, wound healing and angiogenesis [61,62]. However, recent studies havedemonstrated that elastin peptides were able to influence nitric oxide production bycardiomyocytes and ECs [63], offering a potential cardioprotective role for matrikines.

CytokinesMany cytokines are involved in cardiac development and physiology, especially incardiopathologies [64]. In both humans and rodents, circulating and myocardial levels ofinflammatory cytokines such as IL-6 and TNF-α are increased following cardiac injury or inresponse to a developmental defect [64,65]. We have recently found that loss of IL-6 resultsin numerous cardiac abnormalities, including an increased number of fibroblasts and interstitialcollagen deposition, reduced capillary density, increased periostin expression and overall heartdilatation [53]. These and many other studies not only advocate the importance of cytokinesignaling in cardiac function, but illustrate the cellular and biomolecular interrelationships inthe heart, which includes the myocardial ECM.

Perspectives and ConclusionsThis short review is not entirely comprehensive with regards to cell communication and ECMcomposition in the heart. Additional factors that affect cell-cell and cell-ECM interactionsinclude contributions of the CNS, cytoskeletal rearrangements, osteopontin, hyaluronin,SPARC, RANKL, other activators of the PI3K/JAK/ERK/MAPK pathways, different diseaseand physiological states, and a host of other growth factors, hormones and cytokines (otherreviews within this issue cover many of these topics).

Care should be taken when interpreting studies using different techniques (i.e. in vitro matrices,surgically-induced hypertrophy models and knockout models) because of the extremesensitivity and interrelationships involved with cell-cell communication and cell-ECMremodeling; however, interdisciplinary approaches are necessary for properly studying thesesystems. Knockout animal models will continue to elucidate the roles of specific biomolecularinteractions and signals in cardiac function. Studies utilizing knockout animals should includesome sort of rescue of function, as well as tissue-specific and conditional or induced knockouts,so that developmental, pathological and acute dysfunctions can be thoroughly examined. Invitro systems are becoming more advanced to include testable 3-D biochemical and mechanicalinteractions and relationships observed in the in vivo environment, but there is room for

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improvement. Advances in microprinting and tissue bioengineering provide a tremendousopportunity for further development of in vitro ECM systems. Microprinting employs the“printing” of materials onto a substrate, just as an inkjet prints on paper [see 66]. In fact,heterocellular aggregates, which would constitute a part of the organ such as the vasculature,have already been created [66]; the printing of entire organs is not far from being a reality. Theconcepts and techniques used for the microprinting of organs are directly applicable to thecreation of an artificial in vivo-like microenvironment. For example, Kit Parker’s group hasbegun to use a “printed” matrix to examine changes in myocyte shape and size in response togeometric cues from the ECM substrate [67]. Current limitations of cell-ECM in vitro systemsarise from difficulties in obtaining pure cell populations, isolating specific components of theECM and maintaining in vivo cellular and extracellular morphology. The microprinting ofbiological materials could greatly attenuate these issues. The creation of “custom” matricesthat include specific, printed ECM components could model virtually any physiological systemand would be a huge advance for biomedical research. Alternatively, different populations ofliving cells could be printed in a controlled fashion, and induced to produce an ‘authentic’ECM. Any printed ECM matrix would not only include chemical signaling factors of interest,but would also be subjected to mechanical and electrical stimulation, as they could easily bemonitored and manipulated via computer. Moreover, these technical advances could helptransition from a “3D” to a “4D” matrix that includes the variable of time, which would provideadditional models for the in vitro examination of developmental systems, remodeling followingpathological insult and gradual physiological changes in response to disease. These variousmicroenvironments would facilitate the examination of individual and collaborative cellularresponses to a variety of chemical, mechanical and electrical signaling events.

The communication and organization within the cellular environment of the heart has a widerange of clinical applicability, which provides opportunities for a host of additional basic andclinical research. As we have discussed, communication between cardiac cells and the ECMare critical for normal development, homeostasis and response to pathological stress.Moreover, chemical, mechanical and electrical contributions to cell-cell and cell-ECMphysiology are highly interdependent. Continuing to develop these avenues of research willallow for the targeting of specific molecules for pharmacological intervention in the preventionand treatment of a variety of cardiac pathologies, as well as a host of other biological processes.This research should provide further insight into the dynamic regulatory nature of the ECMand also promote the development of novel therapeutics.

AcknowledgmentsThe authors would like to thank Arti Intwala for technical support, helpful suggestions and critical reading of themanuscript. We would also like to thank Dr. Tom Borg for his helpful suggestions. This work was supported byNational Heart, Lung and Blood Institute Grant 1RO1-HL-85847.

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Figure 1.Schematic of cellular changes (grey boxes) and ECM factor expression (black boxes) duringvarious physiological cardiac states (white boxes).

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Figure 2.Representative diagram of the cellular milieu within the myocardium. Note both individualand overlapping contributions of prominent matrix components by each cell type.

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Figure 3.The process of blood vessel formation. To initiate new capillary formation, endothelial cellsof existing vessels must degrade the underlying basement membrane and invade into theneighboring tissue. These processes require the activities of urokinase-plasminogen activator(uPA) and matrix metalloproteinases (MMPs).

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